Intranasal administration of trehalose reduces α-synuclein oligomers and accelerates α-synuclein aggregation

Abstract Abnormal α-synuclein (αSyn), including an oligomeric form of αSyn, accumulates and causes neuronal dysfunction in the brains of patients with multiple system atrophy. Neuroprotective drugs that target abnormal αSyn aggregation have not been developed for the treatment of multiple system atrophy. In addition, treating diseases at an early stage is crucial to halting the progress of neuronal damage in neurodegeneration. In this study, using early-stage multiple system atrophy mouse model and in vitro kinetic analysis, we investigated how intranasal and oral administration of trehalose can improve multiple system atrophy pathology and clinical symptoms. The multiple system atrophy model showed memory impairment at least four weeks after αSyn induction. Behavioural and physiological analyses showed that intranasal and oral administration of trehalose reversed memory impairments to near-normal levels. Notably, trehalose treatment reduced the amount of toxic αSyn and increased the aggregated form of αSyn in the multiple system atrophy model brain. In vitro kinetic analysis confirmed that trehalose accelerated the aggregate formation of αSyn. Based on our findings, we propose a novel strategy whereby accelerated αSyn aggregate formation leads to reduced exposure to toxic αSyn oligomers, particularly during the early phase of disease progression.


Behavioural analysis
To assess the memory of the multiple system atrophy (MSA) model mice, they were subjected to an object-location memory task test 1, 2 consisting of three parts: familiarisation, an acquisition trial, and a test trial.During these three parts, the time spent exploring each object was recorded using a video tracking system (CaptureStar, CleverSys Inc., Reston, VA, USA).Briefly, the mice were placed in a box for 1 h for habituation.Next day, they were placed in the same box containing two objects (O1 and O2) located in the upper-left (O1) and upper-right (O2) corners.The mice explored O1 and O2 for 5 min (acquisition period).After the acquisition period, O2 was moved to the lower right corner (O2 [LR]).The mice explored O1 and O2 (LR) for 5 min (test period).
Mice normally have a spontaneous tendency to spend more time exploring a 'new' object [O2 (LR)] than the original object.The exploration rate (%) was calculated as follows: [mouse interaction time (s) with O2 (LR) / mouse interaction time with O1 + O2 (LR)] × 100.A high exploration rate indicated good memory function.The exploration of an object was defined as positioning the nose towards an object at a distance of < 1 cm and/or touching the object with the nose.Turning around or sitting on an object was not considered exploration.The mouse behaviour was analysed from the recorded images by trained observers blinded to the treatment conditions.
Measurement of trehalose by liquid chromatography-tandem mass spectrometry (LC-MS/MS) 2% sodium dodecyl sulphate (SDS)/8M urea was added to the excised mouse whole brain at 0.1% w/v.The brain was homogenised by sonication and centrifuged at 15,000 g and 4°C for 10 min.The supernatant was assayed for protein by bicinchoninic acid assay and the concentration was adjusted to 12 mg/ml.0.2 ml of the lysate was mixed with 1 ml of 80% acetonitrile containing 2 mM ammonium acetate and centrifuged at 15,000g and 4°C for 10 min, and the resulting supernatant was subjected to LC-MS/MS, a highperformance liquid chromatography (HPLC) system (ExionLC AD, AB SCIEX) coupled to a QTRAP6500+ mass spectrometer (AB Sciex) in electrospray ionization mode.
Trehalose was analysed in negative mode.Ten microlitres of the samples were injected into a high-performance carbohydrate column (250 mm length, 4.6 mm inner diameter, 4 μm particle, Waters) at 35 °C and eluted with an isocratic elution of 80% acetonitrile containing 2 mM ammonium acetate.Mass spectrometry settings are as follows: curtain gas, 30; ion spray voltage, -4500 V; temperature, 300°C; ion source gas 1, 50 psi; ion source gas 2, 60 psi; CAD gas, 7 psi; input potential, -10 V.The flow rate was set at 1.0 ml/min.Trehalose was identified and quantified using an external standard and the optimised multiple reaction monitoring setting of 340.9 m/z and 178.9 m/z for the precursor ion (Q1) and product ion (Q3), respectively.The declustering potential (DP), collision energy (CE) and collision cell exit potential (CXP) were set to -160 V, -18 V and -17 V respectively.For hexose measurements Q1, Q3, DP, EP and CXP were set to 178.8 m/z, 88.9 m/z, -85 V, -10 V and -11 V respectively.
Stimulation for fEPSPs was delivered at a rate of 1 stimulus every 20 s.The stimulation intensity was sufficient to evoke fEPSPs that were 30% of the maximum fEPSP slope (10-90%).To induce LTP, theta burst stimulation was applied 15 min after the start of recording.To assess LTP, Mobius software (Alpha MED Scientific) was used for offline data analysis of the fEPSP slope.The magnitude of LTP was measured as a percentage of the baseline fEPSP slope during the 15 min immediately before theta burst stimulation.
For statistical analysis of the magnitude of LTP in the three groups, the average fEPSP slope of the last 15 min was compared using one-way ANOVA.

Pathological analysis
The mice were anaesthetised with isoflurane (1119, Pfizer, New York, NY, USA).Mice were transcardially perfused with phosphate buffer.Then, the brain was removed and the right hemisphere was fixed with 4% paraformaldehyde for 48 h.After dehydration using a graded ethanol series, the right hemisphere was embedded in paraffin and cut into 4μm-thick sections.
For immunohistochemistry, the sections were dehydrated and pre-treated with heat retrieval using an autoclave for 10 min in 10 mM citrate buffer (pH 6.0).The sections were then subjected to immunohistochemical processing using the avidin-biotinperoxidase complex method (PK-6100; Vector Laboratories, Burlingame, CA) with diaminobenzidine (Sigma-Aldrich, St Louis, MO) and counterstained with haematoxylin.

Immunoblot analysis
The brain samples collected from the left hemisphere were weighted and homogenised with 20 volumes of buffer containing 4% SDS (75 mM Tris-HCl, pH 6.8), 25% glycerol, and 5% β-mercaptoethanol.The samples were passed through a 23-gauge needle attached to a 1-ml syringe and centrifuged at 12,000 rpm for 3 min.
The samples were resolved on an SDS-PAGE gel: 5-20 % Tris-glycine gradient gel (HER-R520L, ATTO, Tokyo, Japan) using an electrophoresis system (AE-6530M, ATTO) under constant current (50 mA for 1 h) and then transferred to a methanolactivated polyvinylidene difluoride membrane (IPVH00010, EMD Millipore, Burlington, MA) using Mini Trans-Blot cell system (1703930JA, Bio-Rad, Hercules, CA) under constant voltage (90 V, 1 h) at 4 °C.For the detection of α-synuclein and phosphorylated α-synuclein, the membranes were immersed in 0.4 % paraformaldehyde in 0.01 M phosphate-buffered saline overnight at 4 °C before membranes were blocked.The membranes were blocked for 1 h at 25 °C with blocking buffer [20 mM Tris-HCl, pH 7.5, 133 mM NaCl and 0.1 % Tween 20 (TBS-T) containing 5 % skimmed milk].The membranes were then washed with washing buffer (TBS-T containing 1 % skimmed milk).After washing, the membranes were incubated with primary antibodies (Supplementary Table 2) for 1 h at 25 °C.The membranes were washed with TBS-T and incubated with HRP-conjugated secondary antibodies (Supplementary Table 2) for 1 h at 25 °C.Signal detection was performed according to the protocol provided by enhanced chemiluminescence (ECL) or ECL prime detection system (RPN2232; RPN2209; GE Healthcare Bioscience, Marlborough, MA, USA).Positive signals were semi-quantified using ImageJ software (NIH).Actin was used for normalisation.

Sample preparation for filter trap assay
Frozen brainstem and hippocampus from MSA model mice were used for filter trap assay.
These samples were aliquoted and frozen at -80°C until used for filter trap assay.

Brainstem
First, the brainstems were weighted and homogenised with 10 volumes of tris-based buffer (30 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol).After homogenization, the samples were passed through a 23-gauge needle and centrifuged at 1,000 x g for 10 min at 4°C [tris-buffered saline (TBS) fraction].Subsequently, the pellet was weighted and two volumes of 2 % triton buffer (30 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 2 % Triton X-100) were added.The samples were passed through a 23-gauge needle and incubated for 30 min at 37 °C with 70 rpm shaking.After incubation, the samples were centrifuged at 14,500 x g for 30 min at 4°C (Triton fraction).Finally, the resultant pellet was sonicated in a volume of 8 M Urea (Urea fraction).

Hippocampus
We collected the TBS fraction as same method for the brainstem.The pellet was weighted and a volume of 2 % triton buffer were added.The samples were passed through a 23gauge needle and incubated for 30 min at 37 °C with 70 rpm shaking.After incubation, the samples were centrifuged at 14,500 x g for 30 min at 4 °C (Triton fraction).
Subsequently, the pellet was weighted and a volume of 2 % of sarkosyl buffer (30 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 2 % N-lauroyl-sarcosine).The samples were passed through a 23-gauge needle and incubated 30 min at 37 °C.After incubation, the samples were centrifuged at 14,500 x g for 30 min at 4 °C (Sarkosyl fraction).Finally, the resultant pellet was sonicated in two volume of 8 M urea (Urea fraction).

Filter trap assay
To detect native Syn, we modified the previously described method of filter trap analysis 3 .
Briefly, fractionated samples were applied to a 0.22-μm cellulose acetate membrane (Advantec, Tokyo, Japan) on a slot blot apparatus (Bio-Rad) using a vacuum manifold.
We washed the membrane with TBS containing 0.1% Tween20, then the membrane was blocked for 1 hour at 25°C using blocking buffer.The subsequent steps were the same as for Western blotting analysis.After signal detection, membranes were stained with Coomassie Brilliant Blue for 10 minutes, and then washed with 10% acetic acid until the background was de-stained.Positive signals were evaluated semi-quantitatively using ImageJ software (NIH) and Coomassie Brilliant Blue-stained bands were used for normalisation.
Then, the sections were washed with Tris-based buffer containing 0.05% Tween-20 and incubated with Duolink ligation reagents for 1 h at 37°C, followed by Duolink amplification reagents for 2.5 h at 37°C.Finally, the sections were washed and incubated with Duolink detection solution for 1 h at room temperature (25°C) and counterstained with haematoxylin.

Cultured cells and treatment
HeLa cells (Japanese Collection of Research Bioresources Cell Bank, Osaka, Japan) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1 % antibiotic and incubated at 37 °C under 5 % CO2.We seeded HeLa cells (cell density: 1.0 x 10 5 cells/dish) on 35-mm dishes and added 2% of maltose or trehalose.

In vitro kinetic assay
An in vitro kinetic assay was performed to detect α-synuclein aggregates according to a previously described protocol 5 .α-Synuclein polymerisation reactions were performed using 40 μM of α-synuclein recombinant protein (Rpeptide, GA; Abclonal, Tokyo, Japan) with 100 mM NaCl and 12.5 mM disaccharide at 37°C and monitored by the fluorescence of 10 μM Thioflavin T (ThT).The samples were agitated every 10 seconds.ThT emissions were measured every 4 min for 48 h at λmax = 477 nm upon excitation at 450 nm.To increase the reproducibility of data between wells, we used one Teflon ball

(
17649-100, Polysciences, Warrington, PA, USA) per well.The plates were sealed with clear polyolefin sealing tape (Thermo Scientific, Waltham, MA, USA) and measured using FlexStation3 (Molecular Devices, Sunnyvale, CA, USA).The photomultiplier gain was set to low (i.e., six flashes per reading), and the signals were read from the bottom of the plate.Transmission electron microscopeRecombinant αSyn (40 μL) was incubated in Tris-based buffer containing NaCl and ThT at 37°C.After glutaraldehyde (final concentration = 2.5%) was added to the samples, they were centrifuged at 15,000 × g for 20 min at 4°C.The samples (5 µL) from different time points were applied to film-coated and carbon-coated copper grids (Nissin EM, Tokyo, Japan) and incubated for 5 min.Excess samples were removed from the edge of the grid using a filter paper.Then, the grids were washed three times with filtered distilled water and blotted onto a filter paper.Negative staining was performed with 2% filtered uranyl acetate for 5 min.Excess dye was blotted onto a filter paper, washed three times with filtered distilled water, and air-dried for 10 min.Photomicrographs were obtained using a JEOL 1230 electron microscope (JEOL Ltd., Tokyo, Japan) at an accelerating voltage of 80 kV.Quantitative analysisPhosphorylated αSyn and human αSyn positive areas were semi-quantified in 5 fields of the olfactory bulb from MSA model mice (N = 5) at x20 original magnification.Olig2 positive areas were semi-quantified using the dentate hilus of MSA model mice (N = 5).Semi-quantitative analyses were performed by the Image J software (NIH, Bethesda, MD).Positive signals were isolated by the colour deconvolution plugin in ImageJ.The images were then converted to binary image and quantified positive signal areas.The number of PLA signals was counted in 4 fields of the olfactory bulb and brainstem from MSA model mice (N = 3) at x40 original magnificent and the average number of these signals was then calculated.